Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

Summary

A series of methods to determine the potential DNRA rate based on ¹⁴NH₄⁺/¹⁵NH₄⁺ analyses is provided in detail. NH₄⁺ is converted into N₂O via several steps and analyzed using quadrupole gas chromatography–mass spectrometry.

Abstract

The importance of understanding the fate of nitrate (NO₃⁻), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO₃⁻ for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO₃⁻ transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the ¹⁵N-labeled ammonium (¹⁵NH₄⁺) accumulation rate in ¹⁵NO₃⁻ added incubation. The determination of the ¹⁴NH₄⁺ and ¹⁵NH₄⁺ concentrations described in this paper is comprised of the following steps. First, the NH₄⁺ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO₃⁻ via persulfate oxidation. Third, the NO₃⁻ is converted to N₂O via an N₂O reductase deficient denitrifier. Finally, the converted N₂O is analyzed using a previously developed quadrupole gas chromatography–mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Introduction

The artificial synthesis of nitrogen fertilizer and its widespread application have greatly perturbed the global nitrogen cycle. It is estimated that the transfer of reactive nitrogen from terrestrial to coastal systems has doubled since pre-industrial times¹. A significant portion of fertilizers applied to a given field is washed away from the soil to rivers or groundwater, primarily as NO₃^{− 2}. This may cause environmental problems such as drinking water pollution, eutrophication, and the formation of hypoxia. NO₃⁻ in water environments is removed from or retained ....

Protocol

1. Preparation of a PTFE envelope for quantitatively capturing gaseous NH₃

1. Place a 60-mm piece of polytetrafluoroethylene (PTFE) tape (25 mm in width) on a small sheet of aluminum foil (approximately 300 mm x 450 mm in size, wiped with ethanol).
2. Ash a glass fiber filter (10 mm in diameter with a pore size of 2.7 μm) at 450 °C for 4 h in a muffle furnace. Place the glass fiber filter a little above the midpoint of the longer axis of the tape (Figure 1a).
3. Spot 20 µL of 0.9-mol/L H₂SO₄ on the center of a GF/D filter, and immediately fold the PTFE tape using two t....

Results

The representative results presented in this paper were derived from ¹⁵N-tracing experiments of salt marsh sediments. The sampled salt marsh was newly created in the aftermath of the 2011 Great East Japan Earthquake in the Moune area of Kesen-numa city in Miyagi Prefecture, Japan. In September 2017, surface sediments (0–3 cm) were collected at two sites in the subtidal and intertidal zones. First, immediately after collection, the sediment was sieved through a 4-mm mesh to remo.......

Discussion

The concentration and isotope ratio of NH₄⁺ for the DNRA analysis was quantified using several methods. The concentrations and isotope ratios of NH₄⁺ are generally measured separately. The NH₄⁺ concentration is typically measured using colorimetric methods including an autoanalyzer^{4,10,15,16,17}........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15N-KNO3	SHOKO SCIENCE	N15-0197
15N-NH4Cl	SHOKO SCIENCE	N15-0034
20 mL PP bottle	SANPLATEC	61-3210-18	Wide-mouth
Aluminum cap	Maruemu	1307-13	No. 20, with hole
Boric acid	Wako	021-02195
Centrifuge	HITACHI	Himac CR21G II
Deoxygenized Gas Pressure & Replace Injector	SANSIN INDUSTRIAL	IP-12
Disposable cellulose acetate membrane filter	ADVANTEC	25CS020AS	Pore size 0.22 µm, 25 mm in diameter
Disposable syringe	Termo	SS-10SZ	10 mL
Disposable syringe	Termo	SS-01T	1 mL
Dulbecco’s Phosphate Buffered Saline (-)	NISSUI PHARMACEUTICAL	5913
Gastight syringe	VICI Valco Instruments	4075-15010	Series A-2, 100 µL
GC/MS	shimadzu	GCMS-QP2010ultra
GF/D	Whatman	1823-010	10 mm in diameter
Glass vial	Maruemu	0501-06	20 mL
Gray butyl rubber stopper	Maruemu	1306-03	No.20-S
H2SO4	Wako	192-04696	Guaranteed Reagent
K2S2O8	Wako	169-11891	Nitrogen and Phosphorus analysis grade
KCl	Wako	163-03545	Guaranteed Reagent
KNO3	Wako	160-04035	Guaranteed Reagent
NaOH	Wako	191-08625	Nitrogen compounds analysis grade
NH4Cl	Wako	017-02995	Guaranteed Reagent
Plastic centrifuge tube	ASONE	1-3500-22	50 mL, VIO-50BN
Pseudomonas chlororaphis subsp. aureofaciens	American Type Culture Collection (ATCC)	ATCC 13985	Freeze-dried, the type strain of Pseudomonas aureofaciens
PTFE sealing tape	Sigma-Aldrich	Z221880	25 mm in width
Reciprocating shaker	TAITEC	0000207-000	NR-10
Screw-cap test tube	IWAKI	84-0252	11 mL
PTFE-lined cap for test tube	IWAKI	84-0262
Tryptic Soy Broth	Difco Laboratories	211825